Mass spectrometry was used to identify scorpion toxin polypeptides that strongly bound to the KcsA channel. Venom from Leiurus quinquestriatus hebraeus was introduced onto an affinity column containing mutant KcsA. Several key mutations were made in the KcsA to make it more closely resemble the eukaryote Shaker potassium channel and permit it to bind strongly to scorpion toxins. MALDI-MS was able to quickly distinguish toxins from whole scorpion venom that bound avidly to the mutant channel from the non-specific binding toxins. The mass accuracy allowed for identification of five toxins, four that are known to be present in the scorpion, and a fifth, an unknown that may represent a newly discovered toxin. These findings ultimately lead to a correlation between the structure of prokaryotic and eukaryotic potassium channels.